Therapeutic agents containing cannabis flavonoid derivatives targeting kinases, sirtuins and oncogenic agents for the treatment of cancers

ABSTRACT

An embodiment of the invention provides a cannabis-based flavonoid pharmaceutical composition including any one or more selected, from among the group of Apigenin, Cannflavin. A. Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin (+)-Taxifolin, Vitexin, and Isovitexin, or their synthases, for the prevention and treatment of certain cancers that can be treated by therapeutically targeting oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and BCL-2. Some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, pancreatic, prostate, leukemia, melanoma as well as any other cancers that overexpress the oncogenic factors inhibited by the cannabis flavonoids identified herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application derives priority from U.S. Provisional PatentApplication 62/156,228 filed 2 May 2015, and is a continuation-in-partof U.S. patent application Ser. No. 14/835,198 filed 25 Aug. 2015.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to flavonoid derivatives and, moreparticularly, to cannabis flavonoid derivatives or the pharmaceuticallyacceptable salt thereof that may be used in a pharmaceutical compositionfor preventing and treating cancer.

2. Description of the Background

Flavonoids are common constituents of plants and cover a wide range offunctions including acting as yellow pigments in petals and leaves toattract pollinating insects. They might also appear as bluish pigments(anthocyanins) to receive certain wavelengths of light, which permitsthe plant to be aware of the photoperiod. Many of these flavonoids alsoprotect the plants by being involved in the filtering of harmfulultraviolet light. Some flavonoids play crucial roles in establishingsymbiotic fungi, while at the same time they fight infections caused bypathogenic fungi.

Flavonoids have relevant pharmacological activities such as:antioxidant, antidiabetic, anti-inflammatory, antiallergic, antibiotic,antidiarrheal, CNS and against cancer.

Cannabis is credited to have several beneficial pharmacologicalproperties. Unfortunately much attention on Cannabis is focused on itsrecreational use as a psychoactive drug. Studies have identified overtwenty flavonoids in the Cannabis plant, such as: cannflavin A,cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside,vitexin, isovitexin, apigenin, kaempferol, myricetin, quercetin,luteolin, homoorientin and orientin. Turner, C. E., Elsohly, M. A., &Boeren, E. G., “Constituents of Cannabis Sativa L. XVII., A review ofthe natural constituents”, Journal of Natural Products, 43(2), 169-234(1980). The distribution of these flavonoids in the plant variesdepending on the type of flavonoid. The total content of flavonoids inthe Cannabis' leaves and flowers can reach 1-2.5% of its dry weightdepending on environment factors and the variety of the plant. It isnoteworthy to mention that even though cannflavin A has been isolatedfrom other plant sources, it is only cannabis that has been shown toharbor all three natural cannflavins.

Cannabis flavonoids have been shown to have several pharmacologicalproperties especially the most common flavonoids such as quercetin,apigenin, luteolin and kaempferol. ElSohly, M. A., Slade, D., “LifeSciences”, 78(5), 539-548 (2005). Chemical constituents of marijuana:the complex mixture of natural cannabinoids). These more commonflavonoids can be found in many other plants and as such are not uniqueto cannabis. Apart from the specific pharmacologic propertiesidentified, cannabis flavonoids are thought to play synergistic roleswith other metabolites in the plant. For example, some flavonoids arevolatile, lipophilic, permeate membranes, and seem to retainpharmacological properties in cannabis smoke. Sauer, M. A., Rifka, S.M., Hawks, R. L., Cutler, G. B., & Loriaux, D. L., “Journal ofPharmacology and Experimental Therapeutics”, 224(2), 404-407 (1983).Marijuana: interaction with the estrogen receptor. Flavonoids maymodulate the pharmacokinetics of THC, via a mechanism shared by CBD, theinhibition of P450 3A11 and P450 3A4 enzymes. These two related enzymesmetabolize environmental toxins from procarcinogens to their activatedforms. P450-suppressing compounds as such serve as chemoprotectiveagents, shielding healthy cells from the activation of benzo[α]pyreneand aflatoxin B1. Offord, E. A., Macé, K., Avanti, O., & Pfeifer, A. M.,“Mechanisms Involved In The Chemoprotective Effects Of Rosemary ExtractStudied In Human Liver And Bronchial Cells”. Cancer Letters, 114(1),275-281, (1997). Benzo[α]pyrene and aflatoxin B1 are two procarcinogenicagents found in cannabis smoke. McPartland, J. M., & Pruitt, P. L.,“Alternative Therapies In Health And Medicine”, 5(4), 57 (1999). Sideeffects of pharmaceuticals not elicited by comparable herbal medicines:the case of tetrahydrocannabinol and marijuana. Cannabis flavonoids thusmay be modulating the therapeutic effects of THC and CBDs by eithersynergistically enhancing desired pharmacologic effects or reducingdetrimental effects. McPartland, J. M., Russo. E. B., “Cannabis AndCannabis Extracts: Greater Than The Sum Of Their Parts?”, Journal ofCannabis Therapeutics, 1(3-4), 103-132 (2001).

There is a small amount of literature on the bioactivity of cannflavinsand other closely related flavonoids isolated either from cannabis orfrom other plants. Barrett et al (1985) reported the inhibitionproperties of cannflavins on prostaglandins with implication oninflammation. Barrett, M. L., Gordon, D., Evans, F. J., “Isolation FromCannabis Sativa L. Of Cannflavin—A Novel Inhibitor Of ProstaglandinProduction”, Biochemical Pharmacology, 34(11), 2019-2024 (1985).

Blanco et al. (2008) reported on cannabidiol and denbinobin and theiruse for the prevention and treatment of gastrointestinal inflammatorydiseases and for the prevention and treatment of gastrointestinalcancers. U.S. patent application Ser. No. 12/681,453 published 2 Sep.2010. Radwan et al. (2008) reported antileishmanial activity forcannflavin A and cannflavin B. Radwan, M. M., ElSohly, M. A., Slade, D.,Ahmed, S. A., Wilson, L, El-Alfy, A. T., Ross, S. A., “Non-CannabinoidConstituents From A High Potency Cannabis Sativa Variety”,Phytochemistry 69(14), 2627-2633 (2008). Brunelli, et al. (2009)reported that isocannflavin B induced autophagy in hormone sensitivebreast cancer cells. Brunelli, E., Pinton, G., Bellini, P., Minassi, A.,Appendino, G., & Moro, L., “Flavonoid-Induced Autophagy In HormoneSensitive Breast Cancer Cells”, Fitoterapia 80(6), 327-332 (2009). Liand Meng (2012) reported the use of the flavonoid Icaritin to treatestrogen receptor related disease. U.S. Pat. No. 8,252,835 issued 28Aug. 2012. Meng et al. (2014) also reported the use of Icaritin to treatcancers. U.S. patent application Ser. No. 14/291,639 published 24 Dec.2014. Apart from the autophagy activity on breast cancer reported byBrunelli and colleagues no other report was seen relating to anticanceractivity of cannflavins. Cytotoxicity studies carried out by the USNational Cancer Institute (NCI) using its 60 cancer cell line panelshowed that cannflavin B was not cytotoxic against cancer cells(NSC:719330).

Despite the foregoing, there has been no prior effort to detail thetargeting of kinases, sirtuins, matrix metalloproteinase, bromodomainsand BCL-2 by the cannflavins or their analogs in the treatment ofcancer. An oncogene is a gene that has the potential to cause cancer. Intumor cells, oncogenes are often mutated or expressed at high levels.Certain cancers overexpress certain oncogenic factors, and theseoncogenic factors may be useful therapeutic targets. However, differentcancer lines respond differently to various oncogenic agents and thatthe response of an outgrowth line to a combination of several suchagents is unpredicatble. The absence of knowledge regarding the use ofcannflavins against these important therapeutic targets prompted thepresent inventors to evaluate the effect of cannflavins against severalkinases, sirtuins, matrix metalloproteinases, bromodomains and BCL-2,with excellent results.

SUMMARY OF THE INVENTION

It is another object to provide a method for the prevention andtreatment of cancer using specific cannabis-based flavonoidpharmaceutical compositions.

It is another object to provide a method for isolating specificcannabis-based flavonoid pharmaceutical compositions from raw plantmaterial that are biologically active in the prevention and treatment ofcancer.

It is still another object to provide a method for synthesizing saidspecific cannabis-based flavonoid pharmaceutical compositions and asynthase.

In according with the foregoing objects, the inventors have successfullysynthesized cannflavins including cannflavin A, cannflavin B andcannflavin C and their derivatives and have demonstrated theiranti-cancer efficacy in various assays including with specific focus onidentifying their therapeutic targets. The present invention relates tothe use of the newly synthesized flavonoids alone or in combination withother bioactive compounds to treat or prevent cancers.

In accordance with the foregoing objects, the present invention providesa flavonoid-based pharmaceutical composition for the prevention andtreatment of cancer having the structure of the general formula shownbelow (see also FIG. 1) or a pharmaceutically acceptable salt thereof.

wherein,

R1-R10 may be any one or more substituents selected from the groupconsisting of a hydrogen molecule (H), a hydroxide molecule (OH), amethyl group comprising one carbon atom bonded to three hydrogen atoms(CH3), an alkoxy group (—OCH3), a carboxyl group (COOH), chlorine (Cl),Bromine (Br), Fluorine (F), Glutamic acid (Glu), geranyl chain, prenylchain and any salts or derivatives of the foregoing. A and B may each beeither a single or double bond.

A method for the prevention and treatment of cancer using the specificcannabis-based flavonoid pharmaceutical compositions is also disclosed,as well as a method for isolating the specific flavonoid-basedpharmaceutical compositions from raw plant material, and a method forsynthesizing said flavonoid-based pharmaceutical compositions.

The present invention is described in greater detail in the detaileddescription of the invention, and the appended drawings. Additionalfeatures and advantages of the invention will be set forth in thedescription that follows, will be apparent from the description, or maybe learned by practicing the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features, and advantages of the present invention willbecome more apparent from the following detailed description of thepreferred embodiments and certain modifications thereof when takentogether with the accompanying drawings in which:

FIG. 1 is an illustration of the general cannabis-based flavonoidpharmaceutical compositions according to the present invention.

FIG. 2 is a flow diagram illustrating a suitable method for isolatingthe specific cannabis-based flavonoid pharmaceutical compositions fromraw plant material.

FIG. 3 is a process diagram illustrating a suitable synthesis approach.

FIG. 4 is an illustration of the specific isolated cannabis-basedflavonoid pharmaceutical compositions including Flavone, Flavanone andFlavanol isolates, according to the present invention.

FIG. 5 is a graphical illustration of the results of the kinaseinhibition by cannflavins presented in Table 1.

FIG. 6 is a graphical illustration of the results of the anticanceractivity presented in Table 3 (Hela cells at A and CMK cells at B).

FIG. 7 is a graphical illustration of the results of the anticanceractivity in mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to preferred embodiments of thepresent invention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

An oncogene is herein defined as a gene that has the potential to causecancer. In tumor cells, oncogenes are often mutated or expressed at highlevels. Certain cancers overexpress certain oncogenic factors includingkinases, sirtuins, bromodomains, matrix metalloproteinases and BCL-2.Thus, these particular oncogenic factors are identified as usefultherapeutic targets for purposes of the present invention.

Given the foregoing targets, some of the cancers that can be treated byuse of cannabis flavonoids based on the inhibition of these therapeutictargets include brain, breast, colon, renal, liver, lung, pancreatic,prostate, leukemia, melanoma as well as any other cancers thatoverexpress the oncogenic factors inhibited

The present invention is a group of cannabis-based flavonoidpharmaceutical compositions selected from among the group of Apigenin,Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin,Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin(Homoorientin), Quercetin, (+)-Taxifolin, Vitexin, and Isovitexin,useful for the prevention and treatment of certain cancers especiallythose that can be treated by targeting kinases, sirtuins, bromodomains,matrix metalloproteinases and BCL-2 which have been identified to beuseful therapeutic targets for some cancers. Some of the cancers thatcan be treated by use of cannabis flavonoids based on the inhibition ofthese therapeutic targets include brain, breast, colon, renal, liver,lung, pancreatic, prostate, leukemia, melanoma as well as any othercancers that overexpress the oncogenic factors inhibited by the cannabisflavonoids identified under this invention.

The cannabis-based flavonoid pharmaceutical composition for theprevention and treatment of cancers has the structure of the generalformula shown below (see also FIG. 1), or a pharmaceutically acceptablesalt thereof.

wherein,

R1-R10 may be any one or more substituents selected from the groupconsisting of a hydrogen molecule (H), a hydroxide molecule (OH), amethyl group comprising one carbon atom bonded to three hydrogen atoms(CH3), an all oxy group (—OCH3), a carboxyl group (COOH), chlorine (Cl),Bromine (Br), Fluorine (F), Glutamic acid (Gin), and any salts orderivatives of the foregoing. A and B may each be either a single ordouble bond.

In an embodiment, a method for the prevention and treatment of cancerusing the specific cannabis-based flavonoid pharmaceutical compositionsabove is also disclosed. Administration may be by various routesincluding oral, rectal or intravenous, epidural muscle, subcutaneous,intrauterine, or blood vessels in the brain (intracerebroventricular)injections. The flavonoid derivatives of the general formula (FIG. 1)according to the present invention and a pharmaceutically acceptablesalt thereof may be administered in an effective dose, depending on thepatient's condition and body weight, extent of disease, drug form, routeof administration, and duration, within a range of from 0.1 to 500 mgbetween 1-6 times a day. Of course, most dosages will be by a carrier.The specific dose level and carrier for patients can be changedaccording to the patient's weight, age, gender, health status, diet,time of administration, method of administration, rate of excretion, andthe severity of disease.

The composition may be formulated for external topical application, oraldosage such as powders, granules, tablets, capsules, suspensions,emulsions, syrups, aerosols, suppositories, or in the form of a sterileinjectable solution. Acceptable carriers and excipients may compriselactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate,calcium silicate, cellulose, methyl cellulose, microcrystallinecellulose, polyvinylpyrrolidone, water, methyl benzoate, propylbenzoate, talc, magnesium stearate, and mineral oil.

Bioactivity

Bioactivity of the above-described compounds were verified and ispresented in Tables 1, 2, and 3 below:

TABLE 1 Kinase FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G IC₅₀ (nM) AuroraA 730 12 >30000 1090 BIKE >20000 >20000 21 >30000 >20000 CK2a 740 76858 >30000 38 CK2a2 350 477 19 >30000 9.7 c-Kit(Y823D) >20000244 >20000 >30000 84 c-Kit(D820Y) >20000 1280 >20000 >30000 113DRAK2 >20000 <1000 980 >30000 >10000 DYRK1/ >20000 <1000 620 >30000 36DYRK1A DYRK1B >20000 1670 6400 >30000 22.8 EFGR(L858R, >20000 >1000680 >30000 >1000 T7790M) EPHB6 >20000 >1000 270 >30000 >1000 FGR >20000224 >20000 >30000 880 FLT3 >20000 41.9 >20000 >30000 44FLT3(D835Y) >20000 12.7 >20000 >30000 45 FLT3(D835V) 220 <1000190 >30000 <1000 FLT3(ITD) >20000 57 >20000 >30000 <1000 FLT4 3309.3 >20000 >30000 4220 (VEGFR3) FMS/CSF1R 1500 199 1200 >30000 4JAK3 >20000 >1000 780 >30000 >20000 KIT 350 <1000 >1000 >30000 <1000KIT(L576P) 180 <1000 >1000 >30000 <1000 KIT(V559D) 200<1000 >1000 >30000 <1000 MELK >20000 232 >1000 >30000 105 MEK5 140 >100084 >30000 >1000 PASK >20000 2060 >20000 >30000 116 PDGFRa — 982 — >300005920 PDGFRa — 0.92 >1000 >30000 2360 (T674I) PDGFRB 3301160 >1000 >30000 3310 PIK3CA >20000 780 >30000 >20000 (1800L) PIK4CB670 >1000 >30000 >20000 PIM-1 >20000 >1000 >30000 78 PIM-3 >20000173 >1000 >30000 35 PIP5K1A >20000 >10000 360 >30000 >20000RIOK1 >20000 >10000 340 >30000 >20000 RIOK3 >20000 >10000280 >30000 >20000 SIK2 >20000 >10000 >1000 >30000 63 SRPK1 >20000 >10000300 >30000 >10000 TNIK >20000 152 >1000 >30000 115

TABLE 2 FBL- FBL- FBL- FBL- FBL- 03A 03B 03C 03D 03G Activity IC₅₀ (μM)SIRT SIRT-1 19.00 27.40 39.50 — — SIRT-2 2.57 10.80 14.00 2.38 24.10SIRT-3 94.90 77.00 65.40 — 66.40 SIRT-5 123.00 104.00 132.00 — 974.00Bromodomain BRD2 NT 9.52 NT NT 12.00 BRD3 NT 7.05 NT NT 8.69 BRD4 NT10.40 NT NT 6.14 Matrix metalloproteinase MMP-2 NT 115.00 NT NT 6.64MMP-3 NT — NT NT 66.30 MMP-7 NT 17.52 NT NT 3.35 MMP-9 NT — NT NT 85.40BCL-2 NT — NT NT 2.49 BCL-XL NT — NT NT —

TABLE 3 FBL- FBL- FBL- FBL- FBL- 03A 03B 03C 03D 03G Cell Line IC₅₀ (μM)A498 (Kidney) 17 NT 14 NT NT A549 (Lung) 17 NT 9.4 NT NT CFPAC-1 12 1712 NT 14.32 (Pancreatic) CMK (leukemia) NT 11.60 NT NT 1.78 COLO-205(Colon) 27 NT 17 NT NT DLD-1 (Colon) 15 NT 13 NT NT HC-1 (Leukemia) NT29.70 NT NT 5.00 HeLa (cervical) NT 10.40 NT NT 2.53 IGROV-1 (Ovarian)29 15 NT NT KMS-11 (Multiple NT NT NT NT NT myeloma) MCF-7 (Breast) 17NT 12 NT NT MiaPaca-2 16 NT 9.5 NT NT (Pancreatic) MOLT-4 NT 13.20 NT NT20.00 (Leukemia) MV4-11 NT 1.43 NT NT 3.1 (Leukemia) NCI-H69 (Small 1611 18 NT 9.5 lung) PC-3 (Prostate) 26 NT 20 NT NT RL (Lymphoma) 5.9 NT12 NT NT SNU-16 (Stomach) NT 15.00 NT NT 4.09 U2-OS (Bone) NT 19.40 NTNT 8.70 UACC-62 27 NT 14 NT NT (Melanoma) U87 (Glioma) 34.00 6.20 12.50NT 5.46

Isolation and Synthesis

A method for isolating the specific cannabis-based flavonoidpharmaceutical compositions from raw plant material is also disclosed.The isolation was realized according to the scheme shown in FIG. 2.

At step 10 an appropriate amount of plant biomass is collected. Forpresent purposes, Cannabis sativa plants were collected by hand. See,Radwan, M. M., ElSohly, M. A., Slade, D., Ahmed, S. A., Wilson, L.,El-Alfy, A. T., Khan, I. A., Ross, S. A., “Non-Cannabinoid ConstituentsFrom A High Potency Cannabis Sativa Variety”, Phytochemistry 69,2627-2633 (2008) and Radwan, M. M., Ross, S. A., Slade, D., Ahmed, S.A., Zulfiqar, F., ElSohly, M. A., “Isolation And Characterization Of NewCannabis Constituents From A High Potency Variety”, Planta Med. 74,267-272 (2008). The collected plant material was air dried under shadeand pulverized into powder.

At step 20 the powder is subjected to supercritical fluid extraction(SFE) by which carbon dioxide (CO²) is used for separating one component(the extractant) from another (the matrix). The extract is evaporated todryness resulting in a green residue.

At step 30, for experimental purposes, a bioassay-guided fractionationwas employed, using a standard protocol to isolate a pure chemical agentfrom its natural origin. This entailed a step-by-step separation ofextracted components based on differences in their physicochemicalproperties, and assessing all their biological activity. The extractedcomponents may, for example, be fractionated by dry column flashchromatography on Si gel using hexane/CH₂Cl/ethyl acetate and mixturesof increasing polarity to yield different fractions. The sample is thendegassed by ultra-sonication to yield an insoluble solid, which solid isthen filtered. The sample may then be subjected to high performanceliquid chromatography (HPLC) using a column Phenomenex Luna™ C18, 5 μm,2×50 mm; eluent, acetonitrile with 0.05% MeOH to confirm the presence ofthe various fractions.

At step 40, bioactivity of the extracts were verified by an anticancercell proliferation assay as described above. This identified thebioactive flavonoids from all the supercritical fluid extracts (SFE). Asreported previously, the identified cannabis-based flavonoid extractsshowed activity against several cancer cell lines including brain,breast, Kaposi sarcoma, leukemia, lung, melanoma, ovarian, pancreatic,colon and prostate cancer.

At step 50 Nuclear Magnetic Resonance Spectroscopy and mass spectrometry(NMR/MS) was performed and the interpreted spectra were consistent withcannabis-based flavonoid compositions as identified above, asillustrated in step 60.

Synthesis Given the known structure of the general formula of FIG. 1 andthe isolate of FIG. 2, a method for synthesizing the same becomespossible. The bioactive cannabis-based flavonoid pharmaceuticalcomposition may be synthesized by the phenylpropanoid metabolic pathwayin which the amino acid phenylalanine is used to produce4-coumaroyl-CoA.

FIG. 3 is a process diagram illustrating a suitable synthesis approachfor the cannflavins. The 2′,4′,6′-Trihydroxyacetophenone was the majorstarting material and the synthesis was carried out using art known tothe industry with modifications yielded the flavonoid backbone whichcontains two phenyl rings. Conjugate ring-closure of chalcones resultsin the familiar form of flavonoids, the three-ringed structure of aflavone. The metabolic pathway continues through a series of enzymaticmodifications to yield the desired Flavone, Flavanone and Flavanol asidentified above and as shown in step 60 (FIG. 3). The specific Flavone,Flavanone and Flavanol isolates are shown in step FIG. 5.

For background see Minassi, A., Giana, A., Ech-Chahad, A., & Appendino,G. “A regiodivergent synthesis of ring A C-prenylflavones”, OrganicLetters 10(11), 2267-2270 (2008). Of course, one skilled in the art willreadily understand that other methods for synthesis are possible, suchas the asymmetric methods set forth in Nibbs, A E; Scheidt, K A,“Asymmetric Methods for the Synthesis of Flavanones, Chromanones, andAzaflavnones”, European Journal Of Organic Chemistry, 449-462.doi:10.1002/ejoc.201101228. PMC 3412359. PMID 22876166 (2012).

Bioactivity Assays

Cannabis flavonoids and their analogs were subjected to kinaseinhibition assay. The compounds were first screened at a singleconcentration of 10 μM in the primary assay. Compounds inhibiting atleast 70% of specific kinases were subjected to further screening todetermine kd/IC₅₀ values. To determine the kd or IC₅₀ values,competition binding assays were established, authenticated and executedas described previously. Fabian et al., “A Small Molecule-KinaseInteraction Map For Clinical Kinase Inhibitors.”, Nat Biotechnol,23(3):329-36, Epub (2005). See also, Karaman et al., “A QuantitativeAnalysis Of Kinase Inhibitor Selectivity”, Nat. Biotechnol. January,26(1):127-32. doi: 10.1038/nbt1358 (2008). For most assays, kinases werefused to T7 phage strains (Fabian, supra) and for the other assays,kinases were produced in HEK-293 cells after which they were tagged withDNA for quantitative PCR detection. In general, full-length constructswere used for small, single domain kinases, and catalytic domainconstructs for large multi-domain kinases. The binding assays utilizedstreptavidin-coated magnetic beads treated with biotinylated smallmolecule ligands for 30 minutes at room temperature which generatedaffinity resins for the kinase assays. The liganded beads were blockedwith excess biotin and washed with blocking buffer (SeaBlock (Pierce),1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reducenon-specific phage binding. Binding reactions were assembled bycombining kinases, liganded affinity beads, and test compounds in 1×binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Testcompounds were prepared as 40× stocks in 100% DMSO and diluted directlyinto the assay (Final DMSO concentration=2.5%). All reactions wereperformed in polypropylene 384-well plates in a final volume of 0.04 ml.The assay plates were incubated at room temperature with shaking for 1hour and the affinity beads were washed with wash buffer (1×PBS, 0.05%Tween 20). The beads were then re-suspended in elution buffer (1×PBS,0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubatedat room temperature with shaking for 30 minutes. The kinaseconcentration in the eluates was measured by quantitative PCR. Anillustration of the kinase interaction process is presented below.Kd/IC₅₀ values were determined using a standard dose response curveusing the hill equation. Curves were fitted using a non-linear leastsquare fit with the Levenberg-Marquardt algorithm.

Percent Control (% Ctrl)

The compound(s) were screened at 10 μM and results for primary screenbinding interactions are reported as ‘% Ctrl’, where lower numbersindicate stronger hits in the matrix.

$\%\mspace{14mu}{Ctrl}\mspace{14mu}{Calculation}\;\left( \frac{{{test}\mspace{14mu}{compound}\mspace{14mu}{signal}} - {{positive}\mspace{14mu}{control}\mspace{14mu}{signal}}}{{{negative}\mspace{14mu}{control}\mspace{14mu}{signal}} - {{positive}\mspace{14mu}{control}\mspace{14mu}{signal}}} \right) \times 100$where:test compound=compound submitted by Environmental Health Foundationnegative control=DMSO (100% Ctrl)positive control=control compound (0%/Ctrl)

The results of the kinase inhibition by cannflavins are presented inTable 1 (above) and in FIG. 5. Inhibition of Sirtuins, matrixmetalloproteinase, bromodomains was also confirmed using standardprotocols and the results are present in Table 2.

PDGERb is implicated in a variety of myeloproliferatue disorders andcancers result from translocations that actuate.

PD Flab by fusion with proteins such as TELETV6, H2, CEV14/TRP11,rabaptin 5 and huntington interacting protein 1.

PD Flab is also overexpressed in metastatic medulloblastoma.

PDGERb is involved also in angiogenasis.

Bioactivity of the above-described compounds has been verified by ananticancer cell proliferation assay using the WST-1(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) colorimetric assay by Roche Life Sciences. Anticanceractivity was tested against several standard cancer cell lines includingbrain, breast, Kaposi sarcoma, leukemia, lung, melanoma, ovarian,pancreatic, colon and prostate cancer. Cells were trypsinized and platedinto 96 well plates in 50 μl of media and incubated overnight. The nextday approximately 18 hours after plating, 50 μl of media containing therequired flavonoid-based pharmaceutical composition was added per well.Cells were plated at a density so that 72 hours post drug addition, thecells will be in log phase (500-2000 cells/well). The compounds andextracts were solubilized in Dimethyl sulfoxide (DMSO). The cells areallowed to proliferate for 72 hours 37° C. in humidified atmosphere of5% CO₂. The experiment is terminated using WST-1 (Roche®) 10 μl per welland absorbance is read at 450 nm/690 nm. The effect of drugs on growthis assessed as percent of cell viability. The IC₅₀ values weredetermined from the extract dose versus control growth curves usingGraphpad Prism® software. All experiments were carried out in duplicateand the mean results determined.

The results of the anticancer activity are presented in Table 3 (above)and in FIG. 6, Hela cells shown at (A) and CMK cells at (B). Todemonstrate a proof of concept in vivo, human pancreatic cancerxenograft CFPAC-1 cells implanted on scid mice were treated with FBL-03Band demonstrated significant inhibition of tumor compared to the controlduring 14 days of treatment. The results of the anticancer activity inmice are presented in FIG. 7.

It should now be apparent that the above-described invention provides apharmaceutical composition for the prevention and treatment of diseasewith specific cannabis-based flavonoid compounds selected from among thegroups of Apigenin, Cannflavin A, Cannflavin B, Cannflavin C,Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin,Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin,(+)-Taxifolin, Vitexin, and Isovitexin, a method for the prevention andtreatment of disease using the specific cannabis-based flavonoidpharmaceutical compositions, a method for isolating the cannabis-basedflavonoid pharmaceutical compositions from raw plant material, and amethod for synthesizing said specific cannabis-based flavonoidpharmaceutical compositions.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the claims. In addition, as one ofordinary skill in the art would appreciate, any dimensions shown in thedrawings or described in the specification are merely exemplary, and canvary depending on the desired application of the invention. Manyvariations and modifications of the embodiments described herein will beobvious to one of ordinary skill in the art in light of the abovedisclosure. The scope of the invention is to be defined only by theclaims, and by their equivalents.

STATEMENT OF INDUSTRIAL APPLICABILITY

Certain cancers overexpress certain oncogenic factors including kinases,sirtuins, bromodomains, matrix metalloproteinases and BCL-2, and sothese particular oncogenic factors are useful therapeutic targets.Cannabis flavonoids have been shown to have several pharmacologicalproperties against these oncogenic factors, but different cancersrespond differently to oncogenic agents and that the response to acombination of several such agents is unpredicatable. There would begreat industrial applicability in the use a group of cannabis-basedflavonoid pharmaceutical compositions selected from among the group ofApigenin, Cannflavin A. Cannflavin B, Cannflavin C, Chrysoeriol,Cosmosiin, Flavocannabiside. Kaempferol, Luteolin, Myricetin, Orientin,Isoorientin (Homoorientin), Quercetin, (+)-Taxifolin, Vitexin. andIsovitexin, for the prevention and treatment of certain cancerstreatable by targeting kinases, sirtuins, bromodomains, matrixmetalloproteinases and BCL-2. Some of the cancers that can be treated byuse of cannabis flavonoids based on the inhibition of these therapeutictargets include brain, breast, colon, renal, liver, lung, pancreatic,prostate, leukemia, melanoma as well as any other cancers thatoverexpress the oncogenic factors inhibited by cannabis flavonoids.

We claim:
 1. A method of treating pancreatic cancer, the methodcomprising administering to a patient in need thereof a cannabis-basedflavonoid pharmaceutical composition having a flavone backbone accordingto chemical structure as shown below, or any pharmaceutically acceptablesalt thereof:

wherein R1-R10 may be any one or more substituents selected from thegroup consisting of a hydrogen molecule (H), a hydroxide molecule (OH),a methyl group comprising one carbon atom bonded to three hydrogen atoms(CH3), a methoxy group, a carboxyl group (COOH), chlorine (Cl), Bromine(Br), Fluorine (F), Glutamic acid (Glu) wherein one of R1 or R3 must beeither 3-methyl-2-butenyl or 3,7-dimethyl-2,6-octenyl, and any salts ofthe foregoing, and the carbon-to-carbon A and B bond may each be eithera single flavanone bond or a double flavone bond.
 2. A method oftreating pancreatic cancer, comprising administering to a patient inneed thereof a composition having a specific chemical structure as shownbelow: